Transfection of the .alpha. and .beta. subunit cDNA clones into cultured mammalian cells has characteristically resulted in low gonadotropin expression levels. This has seriously impeded the production of these hormones on a commercial scale.
It is one aspect of the present invention to provide commercially practical methods for the production of such hormones.
While some genes, such as .beta.-globin (1, 2) and immunoglobulin genes (3-5), require introns for optimal mRNA production, other genes, such as thymidine kinase (6), do not. Intron-dependent increases in gene expression can result from either non-transcriptional (e.g. globin genes) or transcriptional (e.g. immunoglobulin genes) mechanisms.
Isolation of the genes which encode the human and bovine common .alpha., FSH.beta., LH.beta., and human TSH.beta. subunits has been reported (8-14). Ramabhadran et al. (15) first described transfection with and subsequent expression of the human alpha subunit cDNA in mouse cells. Several groups have since reported successful expression of dimeric glycoprotein hormones by transfection of cultured mammalian cells. Some of these groups (16, 17) employed cDNA clones while others (14, 18, 19) have used intron-containing cDNA or genomic sequences.
U.S. Pat. No. 4,840,866 and U.S. Pat. No. 4,923,805 describe the use of cDNA clones (without introns) to produce gonadotropins in cultured mammalian cells. While those expression systems yield biologically active molecules, the yield of the transformed mammalian cells are generally lower than described.
It is another aspect of the present invention to provide improved expression systems, useful with gonadotropins, which result in higher yield.
Matzuk and Boime (18a) mention that an intron inserted into the coding region of the human .alpha. subunit cDNA improved expression results compared with the use of cDNA clones but provided no data to support that contention or specific description of their methods. In a recent publication, Kaetzel and Nilson (7) reported relatively high levels of bovine LH expression in CHO cells. Their system employed genomic sequences for expression of both the .alpha. and LH.beta. subunits. However, the effect of genomic sequences versus cDNA sequences upon LH expression was not addressed in their paper.
It is yet another aspect of the present invention to obviate the confusion represented by the present state of the art and to provide the critical teaching necessary to derive improved vectors encoding dimeric glycoproteins and production methods utilizing such vectors.
Introns have been linked to increased mRNA accumulation in tissue culture cells for rabbit .beta.-globin (1,2,20), E. coli gpt (20), and mouse DHFR (20, 21). Examples of genes containing introns with enhancer elements which increase transcription are the immunoglobulin genes (3-5), the rat cytochrome c gene (22), and the human pro-.alpha.1(I) collagen gene (23). Introns have also been shown to result in increased transcriptional efficiency in transgenic mice for the following genes: rat growth hormone, mouse metallothionein-I, and human .beta.-globin (24). However, introns have no effect on the expression of these last three genes when they are transfected into cultured mammalian cells.
It has been shown that expression levels can be influenced by different 3' untranslated and polyadenylation regions (24, 25). For example, higher expression levels of a galK marker gene result if the bovine growth hormone polyadenylation region is used for transcription termination rather than the SV40 early or human collagen polyadenylation regions (24).